title: Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.
abstract: A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species.
authors.affiliation: Institute of Agricultural Biology and Biotechnology, Italian National Research Council, Via Bassini 15, Milan, Italy. cremonesi@ibba.cnr.it
authors.forename: Paola
authors.initials: P
authors.lastname: Cremonesi
authors.forename: Laura
authors.initials: L
authors.lastname: Vanoni
authors.forename: Stefano
authors.initials: S
authors.lastname: Morandi
authors.forename: Tiziana
authors.initials: T
authors.lastname: Silvetti
authors.forename: Bianca
authors.initials: B
authors.lastname: Castiglioni
authors.forename: Milena
authors.initials: M
authors.lastname: Brasca
mesh_terms: D002611:Cheese / Q000382:microbiology*; D004269:DNA, Bacterial / Q000302:isolation & purification; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D048249:Streptococcus thermophilus / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D001616:beta-Galactosidase